The human herpesviruses cause a spectrum of clinically significant acute and life-long latent infections, and some can transform cells morphologically and oncogenically. During productive infection, all or nearly all viral genes are expressed, resulting in the synthesis of new infectious virus, cell death, and clinical disease. A better understanding of the mechanisms involved in the regulation of viral gene expression during productive infection should shed light on the disease-producing properties of herpesviruses and may ultimately provide novel approaches to intervention in the herpesvirus life cycle. Immediate-early (IE) viral proteins have been implicated as primary factors in regulating viral gene expression during productive infection. In order to define the functions of four of the five HSV IE proteins, a series of small in-frame mutations will be introduced into coding sequences of the genes specifying ICPs O and 27. The effects of these mutations on the transcriptional and translational programs of the mutants will be assessed. The proteins themselves will be purified and characterized for their abilities 1) to bind to specific DNA sequences in viral promoter, 2) to regulate viral gene expression in an in vitro transcription assay, and 3) to bind to other viral and cellular proteins. The DNA sequences in viral promoters that respond to the regulatory activities of ICPs 0 and 27 will be identified by systematic mutational and functional analysis. Mutations will also be introduced into coding sequences of the genes specifying ICPs 22 and 47, and the effects of splicing on the expression of the genes specifying ICPs 0, 22 and 47 will be investigated by substituting unspliced cDNA copies of these genes for their spliced counterparts in the viral genome. The resulting mutant viruses will be characterized with regard to the parameters of viral gene expression. Taken together, these studies should provide a better understanding of the functions of HSSV-1 IE proteins and the manner in which they mediate viral gene expression.